Project Summary/Abstract Bicuspid aortic valve (BAV) is the most common congenital malformation that affects 1-2% of the general population. It is associated with development of aortopathy that manifests as an ascending thoracic aortic aneurysm (TAA) or aortic dissection. Nearly 45% of patients requiring surgery to replace the ascending aorta because of TAA or aortic dissection have BAV. Unlike other congenital diseases like Marfan, Loeys-Dietz or Ehlers-Danlos syndromes, the genetic alteration causing the aortopathy associated with BAV is unknown, despite its known inheritance. The mechanism predisposing the BAV patient to the associated aortopathy is ill- defined. It is known that TAA formation and the propensity for dissection in the setting of BAV involve degeneration of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and apoptosis of aortic smooth muscle cells (SMCs). Furthermore, BAV SMCs display impaired basal oxidative stress response compared to normal SMCs. However, the interplay between the ECM and SMCs has yet to be elucidated. Therefore we propose these specific aims to determine this relationship: Specific Aim 1: Determine how ROS affects ECM production and degradation by aortic SMCs. Specific Aim 2: Identify how collagen microarchitecture affects ROS-induced SMC proliferation and death. Specific Aim 3: Determine how collagen microarchitecture influences SMC function under oxidative stress. Our study will take advantage of our extensive tissue bank of native aortic specimens from patients with TAV and BAV, with and without aneurysmal disease to create high power studies. We will use innovative techniques like macromolecular crowding to modulate the ECM and multiphoton microscopy. Ultimately, with the completion of the aims, we hope to advance the clinical care of patients with BAVs by: 1) Developing pharmaceutical or other therapeutic options for patients with BAV who are at risk for aneurysmal progression, 2) Delineate the molecular mechanisms that initiates the pathogenesis of BAV, and 3) Improving the rupture risk predictions and recommendations for surgical or medical interventions.